US6365359B1 - Inhibitors of prion formation - Google Patents

Inhibitors of prion formation Download PDF

Info

Publication number
US6365359B1
US6365359B1 US09/439,921 US43992199A US6365359B1 US 6365359 B1 US6365359 B1 US 6365359B1 US 43992199 A US43992199 A US 43992199A US 6365359 B1 US6365359 B1 US 6365359B1
Authority
US
United States
Prior art keywords
prp
prion
gene
negative
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US09/439,921
Other languages
English (en)
Inventor
Stanley B. Prusiner
Fred E. Cohen
Thomas L. James
Kiyotoshi Kaneko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Original Assignee
University of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/868,162 external-priority patent/US5962669A/en
Application filed by University of California filed Critical University of California
Priority to US09/439,921 priority Critical patent/US6365359B1/en
Assigned to REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE reassignment REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KANEKO, KIYOTOSHI, PRUSINER, STANLEY B., JAMES, THOMAS L., COHEN, FRED E.
Application granted granted Critical
Publication of US6365359B1 publication Critical patent/US6365359B1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF CALIFORNIA
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/417—Imidazole-alkylamines, e.g. histamine, phentolamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • This invention relates generally to proteins, functionally equivalent pharmacophores and methods of creating and/or detecting inhibitors of prion formation. Specifically, the invention relates to small molecules, peptides and peptide analogs with the ability to either inhibit prion formation or replication and methods of treating a neuropathology such as a prion-mediated neuropathology.
  • Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not efficiently infect another (e.g., a mouse).
  • the prion diseases represent a variety of neurodegenerative states characterized at the neuropathologic level by the presence of spongiform degeneration and astrocytic gliosis in the central nervous system (DeArmond & Prusiner (1996) Current Topics in MicroBiology and Immunology , 207:125-146). Frequently, protein aggregates and amyloid plaques are seen that are often resistant to proteolytic degradation. The neuroanatomic distribution of the lesions varies with the specific types of prion disease. In humans, sporadic Creutzfeldt-Jakob Disease (CJD) accounts for 85% of all cases. The disease presents in the sixth decade of life with dementia and ataxia.
  • CJD Creutzfeldt-Jakob Disease
  • Familial disease carries a variety of monikers such as Gertsmann-Straussler-Scheinker disease (GSS), familial CJD (fCJD) and Fatal Familial Insomnia (FFI) that relate the precise mutation in the PrP gene to a clinical syndrome (Prusiner & Hsaio (1994) Annals of Neurology , 35:385-395; Parchi, et al. (1996) Annals of Neurology , 39:767-778; Montagna, et al. (1998) Brain Pathology , 8:515-520). Disease typically presents in the fourth decade of life with an autosomal dominant pedigree.
  • GSS Gertsmann-Straussler-Scheinker disease
  • fCJD familial CJD
  • FFI Fatal Familial Insomnia
  • the gene contains a single open reading frame and codes for a protein that is proteolytically processed and glycosylated to form a macromolecule with 219 amino acids, a disulfide bridge, two N-linked sugars and a glycophosphotidyl inositol anchor that is exported to the cell surface and concentrated in an endocytic compartment known as the caveolar space (Endo, et al. Biochemistry , 28:8380-8 1989); Stahl, et al. Biochemistry 29:8879-84 (1990); Yost, et al. Nature , 343:669-72 (1990); DeFea, et al. J. Biol.
  • the disease causing form of the prion protein is a multimeric assembly substantially enriched in ⁇ -sheet structure (40% ⁇ -sheet, 30% ⁇ -helices as judged by FTIR spectroscopy) (Pan, et al. (1993) Proc. Natl. Acad. Sci. USA , 90:10962-10966).
  • Transgenic mice carrying a sufficiently high number of copies of mutant gene (the human GSS mutation P101L) on the knockout background develop a spontaneous neurodegenerative disease that is faithful to the neuropathologic expectations developed from a study of the human kindreds.
  • Knockout mice carrying a redacted form of the PrP transgene (90-141; 175-231) also develop a prion disease upon inoculation with full length RML prions (Supattapone, et al. (1999) Cell , 96:869-878).
  • the infection process is more efficient with the “mini” RML prion demonstrating that an artificial prion can be created and that replication efficiency demands fidelity at the amino acid sequence level.
  • PrP C is a kinetically trapped intermediate in the folding of PrP Sc (Cohen & Prusiner (1998) Annual Review of Biochemistry , 67:793-819). This kinetic barrier can be reduced by exogenous administration of the PrP Sc template, mutations to the wild type (wt) PrP sequence, or stochastic processes resulting in infectious, inherited, or sporadic prion diseases. Epitope mapping and peptide studies suggest that much of this conformational plasticity is localized to the middle third of this 231 residue GPI anchored glycoprotein with a 22 amino acid signal sequence (Peretz, et al. (1997) J. Mol. Biol ., 273:614-622).
  • Peptide fragments derived from regions of the PrP sequence have been studied extensively (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA , 89:10940-10944; Tagliavini, et al. (1993) Proc. Natl. Acad. Sci. USA , 90:9678-9682; Forloni, et al. (1993) Nature , 362:543-546; Come, et al. (1993) Proc. Natl. Acad. Sci. USA , 90:5959-5963; Zhang, et al. (1995) J. Mol. Biol ., 250:514-526; Nguyen, et al.
  • peptides chosen from the region 90-145 are compatible with ⁇ -helical, irregularly coiled, and ⁇ -sheet rich conformations when characterized under different conditions (Zhang, et al. (1995) J. Mol. Biol ., 250:514-526).
  • catalytic amounts of ⁇ -sheet rich peptides can facilitate the conformational conversion of peptides with distinct structures into ⁇ -sheet rich isoforms (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA , 89:10940-10944; Nguyen, et al. (1995) Biochemistry , 34:4186-4192).
  • BSE bovine spongiform encephalopathy
  • Molecules are disclosed that interact with the cellular components involved in conversion of PrP C to PrP Sc .
  • the molecules disclosed can be small molecules, peptides or protein analogs, e.g. analogs of PrP C .
  • these molecules interfere with prion formation and/or replication, e.g. by preventing interactions of proteins involved in a prion complex or by interfering with ⁇ -sheet formation.
  • the molecules of the invention promote PrP C conversion to PrP Sc , e.g. by binding to PrP C and facilitating a conformational change from PrP C to PrP Sc .
  • the molecules may be designed to be species specific, meaning that the molecule will only bind to PrP C or Prion Protein Modulator Factor (PPMF) of the same or a genetically similar species.
  • the molecules of the invention may be designed to bind to PrP C or PPMF of genetically a diverse species, i.e. the molecules will not be limited by the “species barrier” that normally limits prion infectivity.
  • the invention features a pharmacophore (defined here as a compound corresponding to a geometric and chemical description of a molecular structure or collection of molecular structures) characterized by an ability to modulate conversion of PrP C to PrP Sc in vivo.
  • the pharmacophore can be a peptide or a small molecule with the ability to bind to PPMF and/or PrP C .
  • the structure of the pharmacophore can be defined by a tertiary surface reflecting the negative image of PPMF at its PrP binding domain and/or a tertiary surface defined by the positive image of a specific discontinuous epitope of PrP protein that includes a small subset of residues.
  • the pharmacophore structure reflects geometric and chemical positions defined by the relative positions of specific amino acid side chains corresponding to the positions of residues 90-231 of the human PrP protein, and in particular residues 168, 172, 215 and 219 corresponding to the human PrP protein.
  • the pharmacophore can also contain an epitope from PPMF that binds to PrP.
  • An object of the invention is to provide an ex vivo system for studying the structural events occurring in conversion, where the system is a cell line treated with a small organic molecule or a peptide that is able to mimic the chemical and geometric features of proteins involved in prion complexing.
  • An advantage of the present invention is that infectivity of prions in a sample can be determined rapidly.
  • the pharmacophore is any one of a collection of molecules that repress prion infectivity and or progression of prion-mediated disease.
  • Any pharmacophore of the invention may inhibit initial infectivity, conversion of PrP C to PrP Sc and/or progression of neurodegeneration by any number of mechanisms, including but not limited to binding a molecule involved in prion complexing, e.g. PrP C or PPMF or inhibiting ⁇ -sheet formation or elongation.
  • Yet another aspect of the invention features a method of repressing conversion of PrP C to PrP Sc , comprising administering an inhibitor that meets the criteria specified by the pharmacophore model.
  • This may be administered prophylactically to a subject at risk of developing a prion-mediated disorder, e.g. a mammal exposed to infectious prions, or to treat a subject that is exhibiting signs of prion-mediated neurodegeneration.
  • a feature of the invention is that the inhibitors can be used to treat subjects suffering from prion-mediated disorders.
  • Yet another aspect of the invention features an assay to identify a PrP pharmacophore, a geometric and chemical specification of a collection of small molecules that could inhibit PrP Sc formation.
  • the assay utilizes the steps of determining functional residues of the PrP protein involved in prion complex interactions, developing three dimensional structures based on these functional residues, comparing the three dimensional structures with a series of compounds having known or calculated tertiary structures, and identifying compounds having a spatial orientation consistent with binding to components of the PrP Sc replication complex (PrP C , PrP Sc , PPMF) at these functional residues.
  • FIG. 1 is a ribbon drawing of the NMR of rSHa PrP(90-23 1). Residues 90-115 are not shown. ⁇ -helical regions are shown in mauve and ⁇ -strands are shown in cyan.
  • FIG. 2 is a model of the putative structure of PrP Sc highlighting the dramatic increase in the ⁇ -sheet structure that has been localized to the region between residues 90-145 by immunologic studies.
  • FIG. 3 illustrates mutations causing inherited human prion disease and polymorphism's in human, mouse and sheep.
  • the x-axis represents the human PrP sequence, with the five octarepeats, the three ⁇ -helical A, B and C and the two ⁇ -strands S1 and S2.
  • Above the line of the human sequence are mutations that cause prion disease.
  • Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotpye of disease.
  • FIG. 4 is an illustration of the distinction between thermodynamic and kinetic models for the energetics of the conversion of PrP C carrying the wild-type (WT) and mutant (MUT) sequences into PrP Sc .
  • ⁇ G is the free energy difference between the PrP C and PrP Sc states and ⁇ G ⁇ is the activation energy barrier separation these two states.
  • ⁇ G WT/MUT is the difference between ⁇ G WT and ⁇ G MUT .
  • the free energy diagrams for the wild-type sequences are shown (solid lines), as are the mutant sequences (broken lines).
  • FIG. 5 is an approach to small molecules in computer screening for inhibitors of PrP Sc replication based on blocking the PrP C :PPMF interaction.
  • FIG. 6 shows dependence of Cp-60 inhibition on (MHM2)PrP Sc concentration using the Time Resolved Fluorescence (TRF) technique of Safar et al (1998).
  • TRF Time Resolved Fluorescence
  • FIG. 7 illustrates the chemical composition of compound 60.
  • FIG. 8 illustrates the chemical structure of analogs based on a substructure search using compound 60.
  • FIG. 8 shows the results of a substructure search of the available chemicals directory with compound 60 as a probe.
  • Nine commercially available compounds have been identified and six have been screened.
  • FIG. 9 is a graphic representation of an open reading frame of the MoPrP and synthetic peptides 89-143 and 89-143, P101L of the MoPrP (MoPrP(89-143)) and MoPrP(89-143, P101L)).
  • the residue corresponding to the mutation is underlined. Confirming that the N-terminal half of the PrP open reading frame is dispensable in prion propagation, PrP27-30 containing G89-S230 can induce prion propagation.
  • HA, HB and HC represent Helices A, B and C, respectively.
  • GPI represents the glycophosphatidyl inositol anchor.
  • PPMF Prion Protein Modulator Factor
  • Prion Protein Modulator Factor is a protein which can be glycosylated and is characterized by binding to PrP C and facilitating a conformational change from PrP C to PrP Sc .
  • the term encompasses any PPMF from any animal allowing for specific differences between different species of animals.
  • the PPMF compounds of the present invention are more particularly characterized herein.
  • pharmacophore is used herein in an unconventional manner.
  • conventional means a geometric and/or chemical description of a class or collection of compounds
  • the term means a compound that has a specific biochemical activity which activity is obtained by the 3-dimensional physical shape of the compound and the electrochemical properties of the atoms making up the compound.
  • the term “pharmacophore” is a compound and not a description of a collection of compounds which have defined characteristics. Specifically, a “pharmacophore” is a compound with those characteristics.
  • pharmacophores of the invention may, for example, mimic or inhibit PrP Sc activity by interaction with (1) the discontinuous epitope on PrP C to which PPMF binds or (2) the surface of PPMF which binds to PrP C .
  • a pharmacophore of the invention has a shape (i.e., the geometric specifications) and electrochemical characteristics substantially as defined by PrP Sc , PPMF, PrP C , or other proteins involved in the prion complex that facilitate the conversion of PrP C to PrP Sc .
  • the term pharmacophore covers peptides, peptide analogs and small molecules.
  • small molecule refers to small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons, which preferably are not comprised of DNA or RNA.
  • treatment used herein to generally mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a prion disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a prion disease or adverse effect attributable to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a cow, pig, sheep, mouse or human, and includes:
  • isolated shall mean separated away from its natural environment.
  • An isolated protein is not necessarily separated away from all materials it is normally present with and may remain glycosylated.
  • corresponding position means the position of an amino acid in a peptide or the position of a codon in a nucleotide sequence corresponding to the same position in the sequence of a different species.
  • amino acid sequence of PPMF also has corresponding positions from one species to another and corresponding positions for four different positions on the discontinuous epitope of PrP C (for five different proteins) are shown in Table 1.
  • FVB refers to a mouse strain commonly used in the production of transgenic mice.
  • PrP mouse prion protein
  • Prnp 0/0 refers to a transgenic animal which has its PrP gene ablated with the “ 0/0 ” indicating that both alleles are ablated whereas “ 0/+ ” indicates only one is ablated.
  • the animal being referred to is generally a transgenic mouse which has its PrP gene ablated i.e., a PrP knockout mouse. In that the PrP gene is disrupted no mouse PrP protein is expressed.
  • CJD sporadic CJD
  • CJD Creutzfeldt-Jakob Disease
  • Iatrogenic CJD refers to disease resulting from accidental infection of people with human prions. The most noted example of such is the accidental infection of children with human prions from contaminated preparations of human growth hormone.
  • Familial CJD refers to a form of CJD which occurs rarely in families and is inevitably caused by mutations of the human prion protein gene. The disease results from an autosomal dominant disorder. Family members who inherit the mutations succumb to CJD.
  • GSS Garnier-Strassler-Scheinker Disease
  • prion shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals.
  • the term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PrP Sc molecules encoded by a PrP gene which expresses PrP C which changes conformation to become PrP Sc .
  • Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats.
  • BSE bovine spongiform encephalopathies
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI).
  • CJD Creutzfeldt-Jakob Disease
  • GSS Gerstmann-Strassler-Scheinker Disease
  • FFI fatal familial insomnia
  • prion includes all forms of prions causing all or any of these diseases or others in any animals used—and in particular in humans and in domesticated farm animals.
  • PrP gene and “prion protein gene” are used interchangeably herein to describe genetic material which expresses PrP proteins, including proteins with polymorphisms and mutations such as those listed herein under the subheading “Pathogenic Mutations and Polymorphisms.” Unless stated otherwise the term refers to the native wild-type gene and not to an artificially altered gene.
  • the PrP gene can be from any animal including the “host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • PrP gene refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein.
  • PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992) and Wopfner et al., J Mol Biol 289:1163-78 (1999), which is incorporated herein by reference to disclose and describe such sequences.
  • the term “genetic material related to prions” is intended to cover any genetic material which affects the ability of an animal to become infected with prions.
  • the term encompasses any “PPMF gene,” “PrP gene,” “artificial PrP gene,” “chimeric PrP gene” or “ablated PrP gene” which terms are defined herein as well as mutations and modifications of such which affect the ability of an animal to become infected with prions.
  • Standardized prion preparations are produced using animals which all have substantially the same genetic material related to prion so that all of the animals will become infected with the same type of prions and will exhibit signs of infection at about the same time.
  • host animal and “host mammal” are used to describe animals which will have their genome genetically and artificially manipulated so as to include genetic material which is not naturally present within the animal.
  • host animals include mice, hamsters and rats which have their endogenous PrP gene altered by the insertion of an artificial gene or by the insertion of a native PrP gene of a genetically diverse test animal.
  • test animal and “test mammal” are used to describe the animal which is genetically diverse from the host animal in terms of differences between the PrP gene of the host animal and the PrP gene of the test animal.
  • the test animal may be any animal for which one wishes to run an assay test to determine whether a given sample contains prions to which the test animal would generally be susceptible to infection.
  • the test animal may be a human, cow, sheep, pig, horse, cat, dog or chicken, and one may wish to determine whether a particular sample includes prions which would normally only infect the test animal. This is done by including PrP gene sequences of the test animal into the host animal, administering PPMF and inoculating the host animal with prions which would normally only infect the test animal.
  • a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster.
  • prions of a given animal will not infect a genetically diverse animal and PPMF of a given animal will not bind to PrP C of a genetically diverse animal.
  • abscessive prion protein gene “disrupted PrP gene,” “ablated PrP gene,” “PrP 0/0 ” and the like are used interchangeably herein to mean an endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative.
  • endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative.
  • nonfunctional PrP genes and methods of making such are disclosed in Büeler, H., et al “Normal development of mice lacking the neuronal cell-surface PrP protein” Nature 356, 577-582 (1992) which is incorporated herein by reference. Both alleles of the genes are disrupted.
  • transgenic or hybrid test animal which develops a prion disease if inoculated with prions which would normally only infect a genetically diverse test animal.
  • the terms are used to describe a transgenic or hybrid animal such as a transgenic mouse Tg(MHu2M) which, without the chimeric PrP gene, would not be susceptible to infection with a human prion (less than 20% chance of infection) but with the chimeric gene is susceptible to infection with human prions (80% to 100% chance of infection).
  • resistant to infection means the animal includes a PrP gene which renders the animal resistant to prion disease when inoculated with an amount and type of prion which would be expected to cause prion disease in the animal.
  • the resistant animals PrP gene includes non-native codons which express amino acids different from those of the native PrP gene which effect the PrP C /PPMF binding site.
  • incubation time shall mean the time from inoculation of an animal with a prion until the time when the animal first develops detectable symptoms of disease resulting from the infection.
  • a reduced incubation time is six months or less, preferable about 100 days ⁇ 25 days or less, more preferably about 30 days ⁇ 10 days or less.
  • MHu2M for a chimeric mouse/human PrP gene wherein a region of the mouse PrP gene is replaced by a corresponding human sequence which differs from mouse PrP at 9 codons;
  • MoPrP Sc for the scrapie isoform of the mouse PrP protein
  • PPMF for Prion Protein Modulator factor in general, i.e., that protein as in any species
  • Prnp 0/0 for ablation of both alleles of an endogenous PrP protein gene, e.g., the Mo PrP gene;
  • PrP CJD for the CJD isoform of a PrP gene
  • PrP Sc for the scrapie isoform of the PrP protein
  • Tg(MHu2M) mice are transgenic mice of the invention which include the chimeric MHu2M gene;
  • Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene.
  • the present invention is based on the discovery that synthetic or isolated molecules (e.g. small molecules, peptides, and the like) with the appropriate tertiary structure have the ability to interact with members of the prion replication complex and to inhibit the formation of de novo protease resistant forms of PrP in appropriate animals.
  • synthetic or isolated molecules e.g. small molecules, peptides, and the like
  • PrP C forms a complex with PPMF and PrP Sc resulting in a ternary complex of PPMF/PrP C /PrP Sc .
  • the complex dissociates due to the lack of affinity of PrP Sc for PPMF.
  • pharmacophores fitting this geometric and chemical description are used to interfere with either the PrP Sc /PrP C or PPMF/PrP C interface.
  • the inhibitors can be used to prevent the initial conversion of PrP C into prions, or later prevent the progression of prion formation.
  • the PrP Sc binding site on the surface of the PrP(90-231) NMR structure appears to form a rather large and discontinuous epitope (Scott, et al., 1997). Accordingly, we have focused on pharmacophores that preferably mimic the PPMF binding site on the surface of PrP C . Identifying pharmacophores of the invention requires the identification of small molecules, peptides, and the like that mimics the positive image of the residues that comprise the PPMF binding site on the surface of the PrP(90-231) NMR structure. A successful compound binds to PPMF, modifying its action, and thereby inhibiting prion replication.
  • deposition diseases such as the prion diseases appear to follow the form:
  • A is the normally synthesized gene product that carries out an intended physiologic role in a monomeric or oligomeric state
  • A* is an conformationally activated form of A that is competent to undergo a dramatic conformational change
  • B is the conformationally altered state that prefers multimeric assemblies
  • B n is the multimeric material that is pathogenic and relatively difficult to recycle.
  • PrP C and PrP Sc correspond to states A and B n where A is largely helical and monomeric and B n is ⁇ -rich and multimeric.
  • B n Two types of kinetic barriers can be imagined that restrict the formation of B n . If the protein in question is relatively large and the conformations of A and B are quite different, then a largely enthalpic kinetic barrier could exist where B can act as a template to reduce the barrier to the conformational change. Alternatively, if a smaller peptide is undergoing the dramatic conformational reorganization (as with ⁇ APP in Alzheimer's disease (Jarrett & Lansbury, Ann N Y Acad Sci . 695:144-8 (1993) or calcitonin in medullary carcinoma of the thyroid or a smaller conformation change is sufficient (as in sickle cell hemoglobin fibril formation (Mirchev & Ferrone, J Mol Biol . 265:475-9 (1997)) the rate limiting step may be the formation of a stable multimeric nucleus (B n ).
  • PrP Sc formation is believed to require an escape from the kinetically trapped monomeric PrP C structure. See FIG. 4 .
  • PrP Sc formation is a first order process where the time from inoculation to disease doubles when the gene dose is changed in animals homozygous and hemizygous for the prion gene. In transgenic animals carrying a mutant gene that causes a spontaneous prion disease, the time to disease is halved when the founder animals are crossed to yield a progeny carrying twice the transgene dose (Cohen & Prusiner, Annu Rev Biochem . 67:793-819 (1998)).
  • the conformational transformation of a monomeric chain follows first order kinetics, while the rate equation for nucleation events follows higher order kinetics dictated by the multimeric state of the nucleus.
  • PrP Sc providing a template to assist the conversion of nascent PrP C molecules implies that some PrP Sc templates should be more efficient at stabilizing the nascent PrP Sc molecule than others in the initial phase of disease propagation. This can be seen in the species barrier to prion transmission where SHa PrP Sc is less efficient than MoPrP Sc in causing disease in mice, and HuPrP Sc is even less efficient than SHa PrP Sc (Scott, et al., Cell 73:979-88 (1993); Telling, et al., Proc Natl Acad Sci US . 91:9936-40(1994); Scott, et al., Proc Natl Acad Sci USA , 94:14279-84(1997).
  • HuPrP Sc provides the most effective inoculum, but it has become clear the BoPrP Sc can cause disease in humans albeit at a much lower frequency (Hill, et al., 1993). There is no evidence that SHaPrP Sc has ever caused disease in humans. It follows that one portion of the molecule is involved in the PrP Sc species specific features of the inoculum while a distinct surface of the molecule is available for interaction with a distinct species specific PPMF molecule.
  • the target size of the infectious particle is 55 kDa, a feature corresponding to a dimer (Bellinger-Kawahara, et al., Virology 164:537-41 (1988)).
  • Identification has been made of four residues that are important to the human PrP C -PPMF interaction: 168, 172, 215 and 219.
  • residues 172, 215 & 219 form a continuous patch on the molecular surface.
  • Residues 170 & 171 are a part of this surface, but mutagenesis experiments demonstrate that they do not participate in this interaction while 168 is clearly not part of this surface in the known structure.
  • Pharmacophores of the present invention take advantage of this conversion process, and preferably have structural aspects that prevent the conversion of PrP C to its conversion competent state (e.g inhibitory pharmacophores).
  • a pharmacophore of the present invention may block PrPC:protein interactions and prevent the conversion process.
  • the PrP pharmacophores of the present invention optionally contain one or more polymorphisms or mutations known to facilitate prion formation.
  • Acidic amino acids should be substituted with basic amino acids and vice versa.
  • Polar amino acids should be substituted with nonpolar amino acids and vice versa. Such mutations may increase efficacy of the pharmacophores for the uses described herein.
  • PrP genes For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:417-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 87:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al., J. Gen. Virol .
  • PrP gene sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991).
  • the PrP gene sequence for mink is published in Kretzschmar et al., J. Gen. Virol . 73:2757-2761 (1992).
  • the human PrP gene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986).
  • the PrP gene sequence for mouse is published in Locht et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986).
  • the PrP gene sequence for sheep is published in Westaway et al., Genes Dev . 8:959-969 (1994).
  • PrP Sc formation is likely to take place in the caveolar space.
  • the inhibitors that follow the pharmacophores of the present invention may be lipidated to increase their efficacy.
  • Pharmacophore inhibitors can be membrane associated by attachment of a covalent linkage to a fatty acid. Prenylation, farnesylation, geranylgeranylation, palmitoylation and myristilation are exemplary modifications that would increase localization of the inhibitor of the invention to the membrane. Linkage to molecules such as cholesterol can also be used to affect localization of the protein.
  • Candidate molecules as inhibitory pharmacophores can encompass numerous chemical classes, including, but not limited to, peptides and small molecules.
  • Candidate pharmacophores can comprise finctional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate pharmacophores often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate inhibitor pharmacophores are also found among biomolecules including, but not limited to: polynucleotides, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate inhibitor pharmacophores can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacologically relevant scaffolds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Inhibitors that modulate molecules involved in prion complex formation and/or PrP Sc conversion can be identified using binding sites on molecules involved in the prion complex, e.g. PrP and PPMF.
  • molecules involved in the prion complex e.g. PrP and PPMF.
  • residues 168, 172, 215 and 219 on the surface of the human PrP C molecule are known to contribute to the integrity of the PrP C -PPMF interface, and thus these molecules define finctional residues on a binding site of PrP.
  • Identification of structural aspects of proteins involved in prion complex formation, such as the side chains involved in the PPMF/PrP C interaction, can define a tertiary structure to be used in an assay to design pharmacophores that modulate molecules and/or protein:protein interactions in the prion complex.
  • a dataset of compounds (small molecules, peptides, etc) having a particular tertiary structure can be identified using techniques known in the art, such as medicinal chemistry, combinatorial chemistry and molecular modeling, to determine molecules that are likely to bind to the atoms or groups of atoms of a protein involved in prion complex formation and/or conversion of PrP C to PrP Sc .
  • factors such as hydrophobicity and hydrophilicity, placement of the functional residues in a structural motif, and mutations involved in prion mediated disorders may also be taken into account.
  • the assay involves (1) matching compounds in a library with the binding site regarding spatial orientation; (2) screening candidate compounds visually using computer generated molecular display software; and (3) experimentally screening actual compounds against PrP C in the presence of PrP Sc to determine compounds which inhibit or enhance conversion of PrP C to PrP Sc .
  • This methods is shown schematically in FIG. 7 .
  • this portion of the molecule can serves as a template for comparison with known molecules, e.g., in a database such as Available Chemicals Database (ACD, Molecular Design Labs, 1997), or it may be used to design molecules de novo.
  • ACD Available Chemicals Database
  • the initial group of identified molecules may contain tens or hundreds of thousands or more of different non-peptide organic compounds.
  • a different or supplemental group may contain millions of different peptides which could be produced synthetically in chemical reactions or via bacteria or phage. Large peptide libraries and methods of making such are disclosed in U.S. Pat. No. 5,266,684, issued Nov. 30, 1993, and U.S. Pat. No. 5,420,246, issued May 30, 1995, which are incorporated herein by reference.
  • Libraries of non-peptide organic molecules are disclosed in PCT publication WO 96/40202, published Dec. 19, 1996, incorporated herein by reference.
  • the initial library of molecules is screened via computer generated modeling, e.g., computer models of the compounds are matched against a computer model of the PPMF binding site on PrP C to find molecules which mimic the spatial orientation and basic polymorphism of PPMF. This screening should substantially reduce the number of candidate molecules relative to the initial group.
  • the screened group is then subjected to further screening visually using a suitable computer program which makes viewable images of the molecules.
  • the resulting candidate molecules are then actually tested for their ability to inhibit PrP Sc formation.
  • a collection of small molecules or peptides can be screened for their ability to affect prion conversion or to mitigate an undesirable phenotype (e.g., a symptom) associated with prion-mediated disease, e.g. neuropathy.
  • the candidate pharmacophores can be screened in either non-transgenic animals or in animals that are transgenic for an alteration in PrP C , and preferably in a transgenic animal with an ablated, endogenous PrP gene, and even more preferably a transgenic animal with an ablated, endogenous PrP gene and an expressed, exogenous PrP gene from a genetically diverse animal that is the target of the pharmacophore, e.g. a PrP 0/0 Tg(MHuM).
  • the candidate pharmacophore is initially tested in an ex vivo cellular array of prion replication optimized using the tools of medicinal chemistry and then administered to a non-human, transgenic animal, and the effects of the candidate determined.
  • the candidate pharmacophore can be administered in any manner desired and/or appropriate for delivery of the small molecules or peptides in order to effect a desired result.
  • the candidate pharmacophore can be administered by injection (e.g., by injection intravenously, intramuscularly, subcutaneously, or directly into the tissue in which the desired affect is to be achieved), orally, or by any other desirable means, and preferably is administered intercerebrally.
  • the in vivo screen will involve a number of animals receiving varying amounts and concentrations of the candidate therapeutic (from no therapeutic candidate to an amount of the candidate that approaches an upper limit of the amount that can be delivered successfully to the animal), and may include delivery of the pharmacophore in a different formulation.
  • the pharmacophore can be administered singly or can be combined in combinations of two or more, especially where administration of a combination of pharmacophores may result in a synergistic effect.
  • the present invention also encompasses pharmaceutical compositions comprising small molecules or peptides fitting the chemical and geometric constraints of the pharmacophores for reducing, inhibiting, or otherwise mitigating plaque formation or prion replication in a subject susceptible to neuronal degenerative disorders associated with protein deposit formation.
  • compositions of the invention preferably contain small molecules or peptides of the present invention. They may also be used in appropriate association with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the small molecules or peptides fitting the chemical and geometric constraints of the pharmacophores can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • the compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.
  • aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.
  • the formulations may also contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compounds can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • the compounds may be formulated with other pharmaceutically active agents, particularly other agents that can modulate onset or symptoms of the condition to be treated.
  • the polycation compound can be co-administered with one or more biologically active agents that reduce protein deposit formation and/or prevent protein deposit formation.
  • biologically active agents include nonsteroid anti-inflammatory drugs (NSAIDs) or aspirin-like drugs (J. R. Vane, Semin Arthritis Rheum 26:2-10 (1997)), selective inhibitors of COX-2 (J. R. Vane Int J Tissue React , 20:3-15 (1998)), protein phosphatases that act on microtubule-associated protein tau protein phosphatases (K.
  • the formulations of the invention have the advantage that they are non-toxic in tested forms of administration.
  • parenteral administration of a solution of the formulations of the invention is preferably nontoxic at a dosage of 0.1 mg/mouse, which is an LD 50 of less than one at 40 mg/Kg.
  • Administration of a compound of the invention may be accomplished by any convenient means, including parenteral injection, and direct intracerebral injection or continuous (e.g., long-term or chronic) infusion.
  • the compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration.
  • the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention.
  • unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host.
  • the implant containing sensitizer is placed in proximity to the site of protein deposits (e.g., the site of formation of amyloid deposits associated with neurodegenerative disorders), so that the local concentration of active agent is increased at that site relative to the rest of the body.
  • the formulations can also be administered by infusion into the brain, and may be administered in either a continuous (e.g., sustained) or non-continuous fashion.
  • Methods, formulations, and devices suitable for delivery to the brain in a continuous (e.g., chronic) or non-continuous (e.g., single, discrete dose per administration) fashion are described in, for example, U.S. Pat. Nos. 5,711,316; 5,832,932; 5,814,014; 5,782,798; 5,752,515; 5,735,814; 5,713,923; 5,686,416; 5,624,898; 5,624,894; 5,124,146; and 4,866,042 (delivery of genetic material).
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the compound for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • the compounds of the invention will generally be administered in dosages of 0.001 mg to 5 mg/kg body weight per day.
  • the range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in animal models (e.g., in the transgenic mice described herein).
  • the mode of administration can have a large effect on dosage.
  • oral dosages in the mouse may be ten times the injection dose.
  • Still higher doses may be used for localized routes of delivery.
  • a typical dosage may be: a solution suitable for intravenous administration; a tablet taken from two to six times daily; or a one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc.
  • the time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.
  • Recombinant PrP Sc was distinguished from endogenous wild-type (wt) Mo PrP Sc by using the SHa/Mo chimeric PrP designated MHM2 that contains a binding site for the anti-SHa PrP 3F4 monoclonal antibody (mAb).
  • the graph theory algorithm of Ullman was employed. This approach has been utilized to aid comparison of protein structures and to search for ligands and sidechain patterns in the Protein Data bank (PDB).
  • PDB Protein Data bank
  • Our algorithm has two main stages. Firstly, the covalent connectivity of the pharmacophore sidechains are compared to the compounds. If at least one match for each of these sidechains is located within the compound, then the distances and angles between these substructures are then compared to the pharmacophore. This approach confers considerable flexibility to the program and facilitates the search for substructures within a 3D pharmacophore that are connected both covalently and non-covalently.
  • Transiently transfected ScN2a cells were incubated with 10 uM KM-00561 for 3 days. Protein-immunoblotting analysis of the lysates were performed before (PK ⁇ ) and after (PK+) proteinase Kup. For immunoblotting, the monoclonal antibody mAb3F4 was used. Compounds were purchased from the appropriate supplier, dissolved in 5% DMSO and incubated with scrapie infected neuroblastoma cells at a variety of concentrations. Administration of 5% DMSO with no compound was used as a negative control, and mevastatin in 5% DMSO was used as a positive control. Experiments showing compounds that present an inhibitory effect on PrP Sc formation in ScN2a cells have been repeated at least 3 times.
  • FIG. 9 shows compound 60 (the original lead compound) and the preliminary screening data at a single concentration for six of the related compounds that are commercially available.
  • Substituted pyridines are a relatively easy target for analog development.
  • the quantitative assay results can be used to determine IC 50 's for each of the analogs in FIG. 10 and then to develop a synthetic plan for building a structure activity relationship.
  • PrP Sc formation is likely to take place in the caveolar space. However, there is no specific reason why the refolded ⁇ -rich peptides should preferentially concentrate in this region. PrP targeted to clatharin coated pits is not converted into PrP Sc . Since PrP is GPI anchored, lipidating the C-terminus of a polypeptide pharmacophore should improve cell localization. Accordingly, the Mo 89-143, P101L peptide was myristylated and applied to cells. The lipidated peptide can then localize to the cell surface and be internalized into endocytic vesicles.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US09/439,921 1997-06-02 1999-11-12 Inhibitors of prion formation Expired - Fee Related US6365359B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/439,921 US6365359B1 (en) 1997-06-02 1999-11-12 Inhibitors of prion formation

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/868,162 US5962669A (en) 1997-06-02 1997-06-02 Nucleic acid encoding prion protein variant
US7660698A 1998-05-12 1998-05-12
US09/439,921 US6365359B1 (en) 1997-06-02 1999-11-12 Inhibitors of prion formation

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US08/868,162 Continuation-In-Part US5962669A (en) 1997-06-02 1997-06-02 Nucleic acid encoding prion protein variant

Publications (1)

Publication Number Publication Date
US6365359B1 true US6365359B1 (en) 2002-04-02

Family

ID=26758283

Family Applications (1)

Application Number Title Priority Date Filing Date
US09/439,921 Expired - Fee Related US6365359B1 (en) 1997-06-02 1999-11-12 Inhibitors of prion formation

Country Status (6)

Country Link
US (1) US6365359B1 (fr)
EP (1) EP0986396A4 (fr)
JP (1) JP2002514224A (fr)
AU (1) AU731291B2 (fr)
CA (1) CA2287848A1 (fr)
WO (1) WO1998055132A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004037854A1 (fr) * 2002-10-23 2004-05-06 Centre For Research And Technology Hellas/Institute Of Agrobiotechnology In.A Sequences peptidiques liant la proteine prion

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2348203B (en) 1998-11-04 2002-06-19 Imp College Innovations Ltd Solube beta-forms of prion proteins, methods of preparation and use
CA2379237A1 (fr) * 1999-07-27 2001-02-01 Imperial College Innovations Limited Matieres vivantes et methodes utiles pour le diagnostic ou le traitement de maladies
IL151758A0 (en) * 2000-03-24 2003-04-10 Univ Massachusetts Prion-free transgenic ungulates
WO2003011016A2 (fr) * 2001-07-31 2003-02-13 Stell Vaches genetiquement modifiees presentant une vulnerabilite reduite a la maladie de la vache folle
IT202000006517A1 (it) * 2020-03-27 2021-09-27 Istituto Naz Fisica Nucleare Small molecules che inducono la degradazione della proteina prionica cellulare

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5025388A (en) 1988-08-26 1991-06-18 Cramer Richard D Iii Comparative molecular field analysis (CoMFA)
WO1991019810A1 (fr) 1990-06-15 1991-12-26 California Biotechnology Inc. Mammifere transgenique non humain presentant la pathologie de formation d'amyloides de la maladie d'alzheimer
WO1993010227A1 (fr) 1991-11-14 1993-05-27 Charles Weissmann Animaux transgeniques exempts de prions
US5434796A (en) 1993-06-30 1995-07-18 Daylight Chemical Information Systems, Inc. Method and apparatus for designing molecules with desired properties by evolving successive populations
US5526281A (en) 1993-05-21 1996-06-11 Arris Pharmaceutical Corporation Machine-learning approach to modeling biological activity for molecular design and to modeling other characteristics

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5750361A (en) * 1995-11-02 1998-05-12 The Regents Of The University Of California Formation and use of prion protein (PRP) complexes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5025388A (en) 1988-08-26 1991-06-18 Cramer Richard D Iii Comparative molecular field analysis (CoMFA)
US5307287A (en) 1988-08-26 1994-04-26 Tripos Associates, Inc. Comparative molecular field analysis (COMFA)
WO1991019810A1 (fr) 1990-06-15 1991-12-26 California Biotechnology Inc. Mammifere transgenique non humain presentant la pathologie de formation d'amyloides de la maladie d'alzheimer
WO1993010227A1 (fr) 1991-11-14 1993-05-27 Charles Weissmann Animaux transgeniques exempts de prions
US5526281A (en) 1993-05-21 1996-06-11 Arris Pharmaceutical Corporation Machine-learning approach to modeling biological activity for molecular design and to modeling other characteristics
US5434796A (en) 1993-06-30 1995-07-18 Daylight Chemical Information Systems, Inc. Method and apparatus for designing molecules with desired properties by evolving successive populations

Non-Patent Citations (79)

* Cited by examiner, † Cited by third party
Title
Baker, H.F., et al. "Aminoacid Polymorphism in Human Prion Protein and Age at Death in Inherited Prion Disease," Lancet (1991) 337:1286.
Barry, R.A., et al., "Monoclonal Antibodies to the Cellular and Scrapie Prion Proteins," J. Infect. Dis. (1986) 154(3):518-521.
Basler et al., "Scrapie and Cellular PrP Isoforms Are Encoded by the Same Chromosomal Gene,"0 Cell, (1986) 46:417-28.
Berger, J.R., et al., "Creutzfeldt-Jakob disease in a physician: A review of the disorder in health care workers", Neurology, (1993) 43:205-206.
Bolton et al., "Identification of a Protein That Purifies with the Scrapie Prion," Science (1982) 218: 1309-11.
Brown et al., "Friendly Fire' in Medicine: Hormones, Homografts, and Cruetzfeldt-Jakob Disease," Lancet (1992) 340: 24-27.
Buchanan et al., "Mortality, Neoplasia, and Creutzfeld-Jakob Disease in Patients Treated with Human Pituitary Growth Hormone in the United Kingdom", BMJ (1991) 302:824-828.
Bueler et al., "Mice Devoid of PrP are Resistant to Scrapie," Cell (1993) 73:1339-1347.
Bueler et al., "Normal Development and Behavior of Mice Lacking the Neuronal Cell-surface PrP Protein," Nature (1992) 356:577-582.
Carlson et al., "Linkage of Protein and Scrapie Incubation Time Genes," Cell (1986) 46:503-511.
Chandler, "Encephaolpathy in Mice Produced by Inoculation with Scrapie Brain Material," Lancet (1961) 1:1378-79.
Cochius et al, "Creutzfeldt-Jakob Disease in a Recipient of Human Pituitary-Derived Gonadotrophin: A Second Case," J. Neurol. Neurosurg. Psychiatry (1992) 55:1094-1095.
Cochius et al., "Creutzfeldt-Jakob Disease in a Recipient of Human Pituitary-Derived Gonadotrophin," Aust. N.Z. J. Med. (1990) 20:592-593.
Cohen, F.E., et al., "Structural Clues to Prion Replication," Science (Apr. 22, 1994) 264:530-531.
Collinge et al., "Genetic Predisposition to Latrogenic Creutzfeldt-Jakob Disease," Lancet (1991) 337:1441-1442.
Cousens, S.N., et al., "Geographical distribution of cases of Creutzfeldt-Jakob disease in England and Wales 1970-84", J. Neurol. Neurosurg. Psychiatry (1990) 53:459-465.
Demaimay et al. 1997. J. Virology 71(12): 9685-9689.* *
Farlie, P.G., et al., "bcl-2 Transgene expression can protect neurons against developmental and induced cell death", Proc. Natl. Acad. Sci. USA (1995) 92:4397-4401.
Gabriel et al., "Molecular Cloning of a Candidate Chicken Prion Protein," Proc. Natl. Acad. Sci. USA (1992) 89:9097-9101.
Gajdusek, D.C., "Unconventional Viruses and the Origin and Disappearance of Kuru," Science (1977) 197:943-960.
Gibbs, Jr. et al., "Creutzfeldt-Jakob Disease Infectivity of Growth Hormone Derived from Human Pituitary Glands," N.Engl. J. Med. (1993) 328:358-359.
Goldfarb et al, "Fatal Familial Insomnia and Familial Creutzfeldt-Jakob Disease: Disease Phenotype Determined by a DNA Polymorphism," Science (1992) 258:806-808.
Goldmann et al., "Different Forms of the Bovine PrP Gene Have Five or Six Copies of a Short, G-C Rich Element within the protein-coding Exon," J. Gen. Virol. (1991) 72:201-204.
Goldmann et al., "Two Alleles of a Neural Protein Gene Linked to Scrapie in Sheep," Proc. Natl. Acad. Sci. USA (1990) 87:2476-2480.
Harris et al., "A Prion-like Protein from Chicken Brain Copurifies with an Acetylcholine Receptor-Inducing Activity," Proc. Natl. Acad. Sci. USA (1991) 88:7664-7668.
Hasty, P., et al., "Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells", Nature (1991) 350:243-246.
Healy et al., "Creutzfeldt-Jakob Disease After Pituitary Gonadotrophins: The Prion is the Problem," BMJ (1993) 307:517-518.
Hecker et al., "Replication of Distinct Scrapie Prion Isolates is Region Specific in Brains of Transgenic Mice and Hamsters," Genes Dev. (1992) 6:1213-1228.
Hsaio et al., "A Prion Protein Variant in a Family with the Telencephalic Form of Gerstmann-Strussler-Scheinker Syndrome," Neurology (1991) 41:681-684.
Hsaio et al., "Inherited Human Prion Diseases," Neurology (1990) 40:1820-1827.
Hsaio et al.,"Linkage of a Prion Protein Missense Variant to Gerstmann-Straussler Syndrome," Nature (1989) 383:342-345.
Kaneko et al. 1995. PNAS 92: 11160-11164.* *
Kaneko, K. et al., "Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation," Proc Natl Acad Sci USA (Sep. 1977) 94:10069-10074.
Kascsak, R.J., et al., "Mouse Polyclonal and Monoclonal Antibody to Scrapie-Associated Fibril Proteins," J. Virol. (1987) 61(12):3688-3693.
Koch et al.,"Creutzfeldt-Jakob Disease in a Young Adult with Idiopathic Hypopituitarism," N. Engl. J. Med. (1985) 313:731-733.
Kretzschmar et al., "Molecular Cloning of a Human Prion Protein cDNA," DNA (1986) 5:315-324.
Kretzschmar et al., "Molecular Cloning of a Mink Prion Protein Gene," J.Gen.Virol. (1992) 73:2757-2761.
Lasmezas et al.,"Recombinant Human Growth Hormone and Insulin-Like Growth Factor I Induce PRP Gene Expression in PC12 Cell," Biochem. Biophys. Res.Commun. (1993) 196:1163-1169.
Locht et al.,"Molecular Cloning and Complete Sequence of Prion Protein cDNA from Mouse Brain Infected with the Scrapie Agent," Proc. Natl. Acad. Sci USA (1986) 83:6372-6376.
Manuelidis et al., "Interspecies Transmission of Creutzfeldt-Jakob Disease to Syrian Hamsters with Reference to Clinical Syndromes and Strain of Agent," Proc. Natl. Acad. Sci USA (1978) 75:3432-3436.
Manuelidis et al., "Serial Propagation of Creutzfeldt-Jakob Disease in Guinea Pigs," Proc. Natl. Acad. Sci. USA (1976) 73:223-227.
McKinley et al, "A Protease-Resistant Protein is a Structural Component of the Scrapie Prion," Cell (1983) 35:57-62.
Medori et al., "Fatal Familial Insomnia, a Prion Disease with a Mutation at Codon 178 of the Prion Protein Gene," N. Engl.J. Med. (1992) 326:444-449.
Muramoto, T., et al., "The Sequential Development of Abnormal Prion Protein Accumulation in Mice with Creuzfeldt-Jakob Disease," Am. J. Pathol. (1992) 140(6):1411-1420.
Nisbet et al., "Creutzfeldt-Jakob Disease in a Second Patient Who Received a Cadaveric Dura mater Graft," J.Am. Med.Assoc. (1989) 261:1118.
Palmer, M.S., et al., "Homozygous Prion Protein Genotype Predisposes to Sporadic Creutzfeldt-Jakob Disease", Nature (1991) 352:340-342.
Pan, K.M., et al., "Conversion of beta-sheets features in the formation of the scrapie prion proteins", Proc. Natl. Acad. Sci. USA (1993) 90:10962-10966.
Pan, K.M., et al., "Conversion of β-sheets features in the formation of the scrapie prion proteins", Proc. Natl. Acad. Sci. USA (1993) 90:10962-10966.
Patel, "France Reels at Latest Medical Scandal," New Scientist, Jul. 31, 1993, p. 4.
Patel, "Placenta Donors to be Screened for Brain Disease," New Scientist, Nov. 20, 1993, p. 10.
Prusiner et al., "Ablation of the Prion Protein (PrP) Gene in Mice Prevents Scrapie and Facilitates Production of Anti-PrP Antibodies," Proc. Natl. Acad. Sci. USA (1993) 90:10608-10612.
Prusiner et al., "Further Purification and Characterization of Scrapie Prions," Biochemistry (1982) 21:6942-50.
Prusiner et al., "Measurement of the Scrapie Agent Using an Incubation Time Interval Assay," Annals. Neurol. (1982) 11(4):353-358.
Prusiner et al., "Molecular Biology of Prion Diseases," Science (1991) 252:1515-1522.
Prusiner et al., "Prion Diseases and Neurodegeneration," Ann.Rev.Neurosci. (1994) 17:311-339.
Prusiner et al., "Transgenic Studies Implicate Interactions Between Homologous PrP Isoforms in Scrapie Prion Replication," Cell (1990) 63:673-686.
Prusiner, S.B., et al., "Immunologic and Molecular Biological Studies of Prion Proteins in Bovine Spongiform Encephalopathy," J. Infect. Dis. (1993) 167:602-613.
Prusiner, S.B., et al., "Prion Protein Biology," Cell (May 1, 1998) 93:337-348.
Prusiner, S.B., et al., "Scrapie Prions Aggregate to Form Amyloid-like Birefringent Rods," Cell (1983) 35:349-358.
Raeber et al., "Attempts to Convert the Cellular Prion Protein into the Scrapie Isoform in Cell-Free Systems," J. Virol. (1992) 66:6155-6163.
Ridley et al., Lancet Occupational Risk of Creuzfeldt-Jakob Disease, (1993) 341:641-2.
Rogers, M. et al., "Epitope Mapping of the Syrian Hamster Prion Protein Utilizing Chimeric and Mutant Genes in a Vaccinia Virus Expression System," J. Immunol. (1991) 147(10):3568-3574.
Scott et al, "Chimeric Prion Protein Expression in Cultured Cells and Transgenic Mice," Protein Sci. (1992) 1:986-97.
Scott et al, "Propagation of Prions with Artificial Properties in Transgenic Mice Expressing Chimeric PrP Genes," Cell (1993) 73:979-988.
Scott, M., et al, "Transgenic Mice Expressing Hamster Prion Protein Produce Species-Specific Infectivity and Amyloid Plaques," Cell (1989) 59:847-857.
Serban, D., et al. "Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins", Neurology (1990) 40:110-117.
Stahl et al., "Glycosylinositol Phospholipid Anchors of the Scrapie and Cellular Prion Proteins Contain Sialic Acid," Biochemistry (1992) 31:5043-5053.
Taraboulos et al., "Regional Mapping of Prion Proteins in Brain," Proc. Natl. Acad. Sci. USA (1992) 89:7620-7624.
Tateishi et al.,"Transmission of Chronic Spongiform Encephalopathy with Kuru Plaques from Humans to Small Rodents," Ann.Neurol. (1979) 5:581-584.
Tateishi, J. and Kitamoto, T., "Developments in Diagnosis for Prion Diseases," Br. Med. Bull. (1993) 49(4):971-979.
Telling, G.C. et al., "Prion Propagation in Mice Expressing Human and Chimeric PrP Transgenes Implicates the Interaction of Cellular PrP with Another Protein," Cell (Oct. 6, 1995) 83:79-90.
Telling, G.C., et al., "Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein," Proc Natl Acad Sci USA (Oct. 1994) 91:9936-9940.
Thadani et al., "Creutzfeldt-Jakob Disease Probably Acquired From a Cadaveric Dura Mater Graft," J. Neurosurg. (1988) 69:766-769.
Valancius, V. and Smithies, O., "Testing and "In-Out" Targeting Procedure for Making Subtle Genomic Modifications in Mouse Embryonic Stem Cells", Mol. Cell Biol. (1991) 11(3):1402-1408.
Westaway et al., "Degeneration of Skeletal Muscle, Peripheral Nerves, and the Central Nervous System in Transgenic Mice Overexpressing Wild-Type Prion Proteins," Cell (1994) 76:117-129.
Westaway et al., Homozygosity for Prion Protein Alleles Encoding Glutamine-171 Renders Sheep Susceptible to Natural Scrapie,: Genes Dev. (1994) 8:959-969.
Wilesmith, J.W., "The epidemiology of bovine spongiform encephalopathy", Acad. Press. (1991) 2:239-245.
Willison et al., "Creutzfeldt-Jakob Disease Following Cadaveric Dura Mater Graft," Neurosurg. Psychiatric (1991) 54:940.
Yehiely, F., et al., "Identification of candidate proteins binding to prion proteins," Neurobiology of Disease (1977) 3(4):339-355.

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004037854A1 (fr) * 2002-10-23 2004-05-06 Centre For Research And Technology Hellas/Institute Of Agrobiotechnology In.A Sequences peptidiques liant la proteine prion
EP1554305A1 (fr) * 2002-10-23 2005-07-20 Centre for Research and Technology Hellas/Institute of Agrobiotechnology Sequences peptidiques liant la proteine prion

Also Published As

Publication number Publication date
EP0986396A4 (fr) 2003-05-02
AU7796798A (en) 1998-12-21
EP0986396A1 (fr) 2000-03-22
WO1998055132A1 (fr) 1998-12-10
CA2287848A1 (fr) 1998-12-10
AU731291B2 (en) 2001-03-29
JP2002514224A (ja) 2002-05-14

Similar Documents

Publication Publication Date Title
Harris Cellular biology of prion diseases
Chiesa et al. Molecular distinction between pathogenic and infectious properties of the prion protein
Jackson et al. Mammalian prion proteins
Moore et al. Prion protein misfolding and disease
Cobb et al. Prion diseases and their biochemical mechanisms
Chiesa et al. Neurological illness in transgenic mice expressing a prion protein with an insertional mutation
Prusiner et al. Genetics of prions
Stahl et al. Prions and prion proteins 1
Safar et al. Prion clearance in bigenic mice
Solomon et al. Prion neurotoxicity: insights from prion protein mutants
Singh et al. Molecular mechanism of the misfolding and oligomerization of the prion protein: current understanding and its implications
Prusiner Prions prions prions
Silva et al. Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion
O’Carroll et al. Prions and Prion-like assemblies in neurodegeneration and immunity: The emergence of universal mechanisms across health and disease
US6365359B1 (en) Inhibitors of prion formation
Mastrianni Prion diseases
Zhang et al. Molecular dynamics studies on the NMR and X-ray structures of rabbit prion proteins
Harris Cell biological studies of the prion protein
Milhavet et al. Effect of Congo red on wild‐type and mutated prion proteins in cultured cells
Kisilevsky et al. Does amyloid enhancing factor (AEF) exist? Is AEF a single biological entity?
DeArmond et al. PrPSc causes nerve cell death and stimulates astrocyte proliferation: a paradox
Hope et al. The scrapie fibril protein and its cellular isoform
US5962669A (en) Nucleic acid encoding prion protein variant
EP1290004A1 (fr) Procede d'identification des points critiques dans des interactions proteines-proteines
Edenhofer et al. Chemistry and molecular biology of transmissible spongiform encephalopathies

Legal Events

Date Code Title Description
AS Assignment

Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, CALI

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PRUSINER, STANLEY B.;COHEN, FRED E.;JAMES, THOMAS L.;AND OTHERS;REEL/FRAME:010673/0923;SIGNING DATES FROM 20000127 TO 20000221

REMI Maintenance fee reminder mailed
FPAY Fee payment

Year of fee payment: 4

SULP Surcharge for late payment
AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF CALIFORNIA;REEL/FRAME:022039/0201

Effective date: 20000912

REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20100402